Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Of the inherited anemias, the hemoglobinopathies, particularly sickle cell disease (SCD) and ?-thalassemia, stand out due to their prevalence and severity. The ability to postnatally elevate fetal hemoglobin (HbF) levels in SCD and ?-thalassemia via co-inheritance of positive genetic modifiers of HbF production or hydroxyurea (HU) treatment can significantly alleviate disease severity. HU, however, has serious side effects and may even be carcinogenic. Novel therapies aimed at elevating HbF expression in adults are, therefore, desperately needed. Three major loci, including BCL11A, modify HbF expression in humans. Together, however, they account for only ~50% of the variation in HbF levels. Hence, significant gaps in knowledge remain regarding the genetic control of HbF production. We will take advantage of two powerful mouse resources to identify novel regulators of ?-like globin switching, the mouse mutant Nan (neonatal anemia) and the newly developed high resolution Diversity Outbred (DO) mapping resource. In Nan a single amino acid change in the second zinc finger of KLF1 (erythroid Krppel-like factor, EKLF) causes sequence selective disruption of binding to a subset of its target genes resulting in severe anemia and a striking failure of hemoglobin switching. Expression of embryonic ?h1 globin is upregulated 100-fold in adult Nan spleen vs. wild type via a BCL11A independent mechanism that is not secondary to stress erythropoiesis. In highly genetically diverse DO mice, expression of ?h1 globin in adults varies substantially from individual to individual. Thus, Nan and DO mice are ideal tools with which to detect novel regulators of ?-like globin switching using powerful, unbiased genetic QTL (quantitative trait locus/loci) mapping integrated with global genomic and proteomic strategies. The overall goal of this proposal is to identify novel genes regulating ?-like globin switching. To accomplish this goal, the specific aims are to (1) map modifiers of embryonic globin expression in Nan F2 intercrosses and in DO mice to identify KLF1-dependent and independent loci, respectively; (2) perform ChIP-seq in erythroid populations to compare DNA targets differentially bound by wild type (WT) and mutant (Nan) KLF1; (3) obtain erythroid global transcriptome (RNA-seq, miR-seq) and phospho-proteome profiles to identify gene expression and post-transcriptional differences; and (4) analyze and integrate all data to identify, prioritize, and initiate functional analysis of the most compelling candidate genes. Identification of genetic loci regulating ?-like globin switching, differences i Nan- vs. WT-KLF1 DNA targets, and differences in the transcriptome and proteome of Nan and WT erythroid cells will converge to identify novel regulators of ?-like globin switching, thereby providing important new therapeutic targets for hemoglobinopathies.